Bacterial Secretion Systems PDF

Bacterial Secretion systems A/Prof Nichollas Scott Scott Lab, The Peter Doherty Institute for Infection and Immunity www.scottnelaboratory.com 01-03-2024 (MIIM30011)  Learning goals At the completion of this...

Bacterial Secretion systems A/Prof Nichollas Scott Scott Lab, The Peter Doherty Institute for Infection and Immunity www.scottnelaboratory.com 01-03-2024 (MIIM30011)  Learning goals At the completion of this lecture students should: Have an understanding of the apparatuses used to transport proteins across bacterial (and host) membranes Be able to describe the different mechanisms of secretion in Gram negative and Gram positive bacteria Provide specific examples of secretion systems and the characteristics of key secretion systems observed within pathogens  Bacterial Secretion Systems: Why are they important? A variety of specialised nanomachines are used for transporting proteins across bacterial membranes We call these nanomachines Secretion Systems Secreted proteins allow bacteria to modulate their surroundings and there are three possible fates for secreted proteins. They can remain associated with the bacterial outer membrane (anchored) Be released into the extracellular space Injected directly into a target cell  Transport across membranes is so important multiple systems have evolved to do it The transport of proteins across membranes appears to have evolved multiple times Some transport proteins cross a single membrane while others traverse multiple membranes (linked to function) Many secretion systems are associated with pathogens and considered virulence factors Secretion systems can be divided into different classes (Example Type 1 to 6*) Green E.R. et al. Microbiol Spectr. 2016 Feb; 4(1): 10.1128/microbiolspec.VMBF-0012-2015. *new classes are discovered all the time with atleast 11 systems known to date!  Transport systems common to Gram Negative and Positive bacteria Some systems are found in both Gram Positive and Negative species Two common systems used to transport proteins across the inner membrane are the: The general secretory pathway (Sec Translocase Pathway) responsible for the secretion of unfolded proteins The Twin arginine transport (TAT) pathway responsible for the secretion of folded proteins Costa T. R.D. et al Nature Reviews Microbiology volume 13, pages343–359 (2015)  Transport across the inner membrane: The Sec Translocase Sec translocation allows the transport of unfolded proteins Sec translocation can be a co- translational or post translational process depending on the protein and how it interacts with sec components (SRP vs SecB) Protein targeted for the Sec system by a signal tag, a 20– 30-residue sequence at the N- termini of proteins Papanikou E, et al. Nat Rev Microbiol. 2007 Nov;5(11):839-51. doi: 10.1038/nrmicro1771.  Transport across the Inner Membrane: The Tat Translocase sytem The Tat system allows the export of folded proteins from the cytoplasm Recognition is mediated by a twin arginine tag at the N-termini of proteins recognised by TatB/C Recognition leads to formation of a TatA pore (the TatA ring) which the Tat protein translocates through Thought experiment: Why would it be advantageous to transport folded proteins? Palmer T and Berks B.C. Nat Rev Microbiol. 2012 Jun 11;10(7):483-96.  Sec and Tat secreted proteins can be associated with virulence Both Sec and Tat secreted proteins can contribute to virulence: Sec example (NamA) autolysins of Listeria monocytogenes contribute to host colonization by remodelling peptidoglycan Tat example Phospholipase C of P. aeruginosa (PlcH) requires Tat based secretion to be functional Lenz et al Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12432-7. doi: 10.1073/pnas.2133653100. Ochsner U.A. et al Proc Natl Acad Sci U S A. 2002 Jun 11;99(12):8312-7. doi: 10.1073/pnas.082238299.  Transport systems found within Gram- Negative bacteria Gram negative species differ from Gram positive species in that they have two membranes (inner membrane / outer membrane) The need to traverse both membranes to secrete proteins requires unique mechanisms (one-step or multi-step mechanisms) These secretion systems can be divided on where they deposit their proteins Into the environment Into a host cells Green E.R. et al. Microbiol Spectr. 2016 Feb; 4(1): 10.1128/microbiolspec.VMBF-0012-2015.  Transport systems which traverse the outer membrane of Gram-Negative bacteria Multiple transport systems have evolved to allow the transport of proteins across the outer membrane from both the cytoplasm and periplasmic space Secretion Secretion Secretion Folded Number of mechanism Apparatus Signal Substrates? Membranes Organisms Type I secretion T1SS C-terminus No 2 E. coli Pseudomonas/ Type II secretion T2SS N-terminus Yes 1 Aceintobacter Type III secretion T3SS N-terminus No 2–3 EPEC, Shigella, Salmonella Type IV secretion T4SS C-terminus No 2–3 Legionella, Coxiella Autotransporter pathway T5SS N-terminus No 1 Neisseria, E. coli No known Pseudomonas, Type VI secretion T6SS secretion signal Yes (Partial) 2–3 Burkholderia Costa T. R.D. et al Nature Reviews Microbiology volume 13, pages343–359 (2015) Green E.R. et al. Microbiol Spectr. 2016 Feb; 4(1): 10.1128/microbiolspec.VMBF-0012-2015.  Transport systems which traverse the outer membrane of Gram-Negative bacteria Multiple transport systems have evolved to allow the transport of proteins across the outer membrane from both the cytoplasm and periplasmic space Secretion Secretion Secretion Folded Number of mechanism Apparatus Signal Substrates? Membranes Organisms Type I secretion T1SS C-terminus No 2 E. coli Pseudomonas/ Type II secretion T2SS N-terminus Yes 1 Aceintobacter Type III secretion T3SS N-terminus No 2–3 EPEC, Shigella, Salmonella Type IV secretion T4SS C-terminus No 2–3 Legionella, Coxiella Autotransporter pathway T5SS N-terminus No 1 Neisseria, E. coli No known Pseudomonas, Type VI secretion T6SS secretion signal Yes (Partial) 2–3 Burkholderia Class Question: From this list which secretion systems transfer proteins into host cells? Costa T. R.D. et al Nature Reviews Microbiology volume 13, pages343–359 (2015) Green E.R. et al. Microbiol Spectr. 2016 Feb; 4(1): 10.1128/microbiolspec.VMBF-0012-2015.  Transport systems which traverse the outer membrane of Gram-Negative bacteria Multiple secretion systems translocate proteins to the environment: 2 examples of these are the T1SS and T5SS which represent one-step and multi-step processes Secretion Secretion Secretion Folded Number of mechanism Apparatus Signal Substrates? Membranes Organisms Type I secretion T1SS C-terminus No 2 E. coli Pseudomonas/ Type II secretion T2SS N-terminus Yes 1 Aceintobacter Type III secretion T3SS N-terminus No 2–3 EPEC, Shigella, Salmonella Type IV secretion T4SS C-terminus No 2–3 Legionella, Coxiella Autotransporter pathway T5SS N-terminus No 1 Neisseria, E. coli No known Pseudomonas, Type VI secretion T6SS secretion signal Yes (Partial) 2–3 Burkholderia Costa T. R.D. et al Nature Reviews Microbiology volume 13, pages343–359 (2015) Green E.R. et al. Microbiol Spectr. 2016 Feb; 4(1): 10.1128/microbiolspec.VMBF-0012-2015.  Secretion into the environment: T1SS E. coli Type I systems are employed in many different species for the secretion of: Toxins/Proteases/Lipases T1SS allow the translocation of unfolded proteins from the cytoplasm directly across the outer membrane without a periplasmic intermediate (employs 3 proteins to form a “channel–tunnel” conduit, one-step mechanism) The secreted proteins lack a typical signal peptide, but instead have a C-terminal secretion signal of ~60 amino acids Spitz et al Microbiol Spectr. 2019 Mar;7(2). doi: 10.1128/microbiolspec.PSIB-0003-2018.  Secretion into the environment: T5SS T5SS allows the translocation of proteins across the plasma membrane after Sec translocation (multi step mechanism) T5SS proteins contain four key elements An N-terminal signal tag An internal passenger domain A linker region A C’ terminal β barrel / translocator domain Insertion of the translocator domain is mediated by the BAM complex within the outer membrane Multiple classes of T5SS exist, some anchor proteins to the surface some allow the release of proteins after cleavage Clarke et al. Front Immunol. 2022 Jul 1;13:921272. doi: 10.3389/fimmu.2022.921272 Costa T. R.D. et al Nature Reviews Microbiology volume 13, pages343–359 (2015)  T1SS and T5SS proteins can be associated with virulence Examples of T1SS & T5SS proteins associated with virulence: T1SS: α-haemolysin HlyA of E. coli T5SS secreted: IgA1 protease of Neisseria Gonorrhoeae T5SS anchored: Ag43 of E. coli Bjakdi S. et al Eur J Epidemiol. 1988 Jun;4(2):135-43. doi: 10.1007/BF00144740. Clarke et al. Front Immunol. 2022 Jul 1;13:921272. doi: 10.3389/fimmu.2022.921272 *Johannsen D.B. et al Infect Immun. 1999 Jun; 67(6): 3009–3013.  Transport systems which translocate proteins into hosts For pathogens subverting the host response can be essential for survival Some Gram-Negative pathogens have evolved dedicated secretion systems which can translocate proteins across 3 membranes (inner/outer/host) These secreted proteins are commonly referred to as ‘Effectors’ Green E.R. et al. Microbiol Spectr. 2016 Feb; 4(1): 10.1128/microbiolspec.VMBF-0012-2015.  Transport systems which translocate proteins into hosts Multiple secretion systems can translocate proteins into host/neighbour cells including the T3SS, T4SS and T6SS Secretion Secretion Secretion Folded Number of mechanism Apparatus Signal Substrates? Membranes Organisms Type I secretion T1SS C-terminus No 2 E. coli Pseudomonas/ Type II secretion T2SS N-terminus Yes 1 Aceintobacter Type III secretion T3SS N-terminus No 2–3 EPEC, Shigella, Salmonella Type IV secretion T4SS C-terminus No 2–3 Legionella, Coxiella Autotransporter pathway T5SS N-terminus No 1 Neisseria, E. coli No known Pseudomonas, Type VI secretion T6SS secretion signal Yes (Partial) 2–3 Burkholderia Costa T. R.D. et al Nature Reviews Microbiology volume 13, pages343–359 (2015) Green E.R. et al. Microbiol Spectr. 2016 Feb; 4(1): 10.1128/microbiolspec.VMBF-0012-2015.  Secretion into hosts: T3SS T3SS are found in a wide range of Gram- negative pathogens including: Salmonella and Yersinia - host cell invasion T3SS directly translocates effectors into the cytoplasm of target eukaryotic cells. T3SS share homologues to flagella biosynthesis and assembly systems T3SS secreted effector proteins contain an N- terminal sequence that is recognised and bound by specific chaperones Chaperones guide their specific secreted proteins to the secretion apparatus Deng et al Nat Rev Microbiol. 2017 Jun;15(6):323-337. doi: 10.1038/nrmicro.2017.20. Portaliou A.G. et al. Trends Biochem Sci. 2016 Feb;41(2):175-189. doi: 10.1016/j.tibs.2015.09.005.  Organisation and trends in T3SS Type III secretion is triggered when a EPEC triggers a characteristic 'attaching and effacing' lesion pathogen comes into contact with a target eukaryotic cell and can be considered “contact-dependent” Genes encoding T3SSs can be either plasmid encoded or encoded within genomic pathogenicity islands. The genes encoding effectors do not have to be genetically linked to the T3SS machinery Some pathogens have multiple T3SS Most Salmonella have two T3SS (called locus of enterocyte effacement (LEE) in E. COLI E2348/69 SPI1 & SPI2) Enteropathogenic E. coli (EPEC) encodes a single T3SS Pearson et al. Annu Rev Genet. 2016 Nov 23;50:493-513. Deng et al. Nat Rev Microbiol. 2017 Jun;15(6):323-337. doi: 10.1038/nrmicro.2017.20.  Secretion into hosts: T4SS T4SS subfamilies T4SS are evolutionarily related to bacterial VirB/VirD conjugation systems Enable the translocation of DNA or proteins to other bacteria as well as eukaryotic cells Two main subfamilies Dot/Icm Conjugation machineries (DNA and protein transfer via the VirB/VirD system-T4ASS) Effector protein translocation systems (Protein transfer via Dot/Icm system T4BSS) Notable VirB/VirD examples the Helicobacter pylori Cag system and Bordetella pertussis pertussis toxin secretion (sec dependent) Alvarez-Martinez C.E. Christie P.J. Microbiol Mol Biol Rev. 2009 Dec;73(4):775-808. doi: 10.1128/MMBR.00023-09. Elizabeth Boudaher E., Shaffer C.L. Medchemcomm. 2019 May 8;10(5):682-692. doi: 10.1039/c9md00076c. Christie P.J et al. Biochim Biophys Acta. 2014 Aug;1843(8):1578-91.  Organisation and trends in T4ASS VirB/VirD The prototypic VirB/VirD system was first characterised in Agrobacterium tumefaciens Functions to deliver a fragment of the A. tumefaciens genome to the host cell (oncogenic T-DNA) plus effector proteins leading to Crown Gall Disease in plants. Delivers DNA via a pilus (VirB2) enabling cell-to-cell transfer between the bacterial and target cell DNA/protein transfer appears to occur in a single step process from the cytoplasm to outside the cell Fronzes et al. Nat Rev Microbiol. 2009 Oct;7(10):703-14. doi: 10.1038/nrmicro2218.  Organisation and trends in T4BSS Dot/Icm Legionella pneumophila & Coxiella burnetii both possess Dot/Icm systems Translocation facilitated by a C- terminal translocation signal Responsible for the translocation of 100s of proteins which block, blunt and subvert host cells Allows the translocation of proteins from the cytoplasm and periplasm Icm stands for: intracellular multiplication and dot for: defective organelle trafficking Essential for replication in the host Kitao T. et al. Microbiol Immunol. 2022 Feb;66(2):67-74. doi: 10.1111/1348-0421.12951.  Secretion into other cells: T6SS T6SS appears to share structural features with bacteriophage cell puncturing proteins Consist of an intracellular contractile and membrane-bound molecular machine Contact-dependent secretion mechanism which act as a “poisoned spear” to deliver effectors Targets both bacteria as well as eukaryotic cells Found in ~25% of Gram-negative bacteria, including human and animal pathogens Prototypic example: P. aeruginosa Species can possess multiple T6SSs for example B. thailandensis has five T6SSs Recently described, not well characterized Coulthurst S. Microbiology. 2019 May;165(5):503-515. doi: 10.1099/mic.0.000789.  T6SS, inter-bacterial combat T6SS can be used as a mechanism Dueling response of P. aeruginosa to attack by V. cholerea to compete with other bacteria (bacterial antagonism) Injection (stabbing) of toxins into neighbouring bacterial cells ‘Attacker’ is protected via cognate immunity proteins* Can also be used as a form of bacterial retaliation (tit for tat phenomena) Currently challenging to predict if a T6SS is anti-Bacterial or anti- “tit for tat” response Eukaryotic, some are both! * If a strain possesses an immunity protein this can block effector function Basler et al Cell. 2013 Feb 14;152(4):884-94. doi: 10.1016/j.cell.2013.01.042.  Transport systems unique to Gram Positive bacteria (T7SS) Unique secretion systems also exist in Gram Positive bacteria such as the Type 7 Secretion system Prototypic example from Mycobacterium tuberculosis (related systems also seen in Staphylococcus aureus) Transports proteins across the inner membrane and the mycobacterial cell wall, transport dependent on a C-terminal signal tag Unknown how effector of T7SSs cross the outer membrane but it is thought they are folded Required for virulence Mycobacteria can have up to five different T7SSs Spencer B.L. and Doran K.S. PLoS Pathog. 2022 Jul 28;18(7):e1010680 Rivera-Calzada A. et al. Nat Rev Microbiol. 2021 Sep;19(9):567-584. doi: 10.1038/s41579-021-00560-5.  Summary Multiple secretion systems exist (T1SS, T3SS, T4SS, T5SS, T6SS & T7SS) Systems differ in their ability to transport folded/unfolded proteins, signal sequence locations (N & C termini) and target location (host & environment) Rapisarda C. et al. Annu Rev Microbiol. 2018 Sep 8;72:231-254.  Summary table Secretion Secretion Secretion Folded Number of mechanism Apparatus Signal Substrates? Membranes Organisms Type I secretion T1SS C-terminus No 2 E. coli Pseudomonas/ Type II secretion T2SS N-terminus Yes 1 Aceintobacter Type III secretion T3SS N-terminus No 2–3 EPEC, Shigella, Salmonella Type IV secretion T4SS C-terminus No 2–3 Legionella, Coxiella Autotransporter pathway T5SS N-terminus No 1 Neisseria, E. coli No known Pseudomonas, Type VI secretion T6SS secretion signal Yes (Partial) 2–3 Burkholderia Mycobacterium, Type VII secretion T7SS C-terminus Yes (dimers) 2 Staphylococcus